Background
Lung cancer is relatively resistant to currently available chemotherapy and radiotherapy regimens. However, observations of lymphocytic infiltrates or generation of cytotoxic T lymphocytes (CTLs) recognizing lung tumor antigens in murine and human tumors [
1‐
5] suggest that an immune reaction could potentially help eliminate tumor cells. While immune effector cells are present, often in high numbers, immunological rejection of tumors is usually not complete, indicating that there may be defects in the generation or execution of an anti-tumor immune response [
6]. Many factors likely contribute to an incomplete immune response including defective processing and presentation of tumor antigens and loss of MHC class I antigen expression [
7], absence of co-stimulation [
8], absence of tumor reactive T cells [
9,
10], alteration in T cell signaling [
11‐
14], presence of a variety of immunosuppressive factors [
15,
16], and inhibition of tumor T cell infiltration [
17,
18]. Clearly the dynamics between a growing tumor and host is very complex and cannot be explained by a single mechanism. Most of these mechanisms are currently studied in murine tumor models. There are relatively few reports about the actual events that occur
in situ between human lung tumors and effector cells of the immune response.
Since the major biological activity against tumors
in vitro and
in vivo displayed by tumor infiltrating lymphocytes (TILs) can be attributed to CD8
+ T cells [
19], adoptive immunotherapy using
in vitro expanded tumor antigen-specific CD8
+ CTLs, has been considered as a feasible option for
in vivo eradication of tumors [
20]. A variety of lymphoid cells that are able to lyse tumor cells have been described [
19‐
24]. Furthermore, in humans the
in vitro generation and expansion of autologous CTLs that recognize lung [
1‐
5], melanoma [
25‐
27], renal [
28], ovarian [
29], leukemia [
30‐
32], lymphoma [
33,
34] and breast [
35‐
38] tumor antigens have now been reported. Since tumor antigen-specific CTLs are effector cells that are known to play an important role in inhibiting tumor growth, it is important to understand the interaction between tumor-specific CTLs and autologous lung tumors. Although there were subcutaneous models that generated to study these interactions [
5], it is also important to examine the interaction
in vivo between these cells and their target tumor grown orthotopically. The difficulties in studying the interplay between a patient's immune response and tumors highlights the need to develop an
in vivo model to study the interaction between these cells in a model which may replicate more closely their environment in patients.
In this study, primary lung tumor cells and tumor antigen-specific CTLs isolated from the original surgical specimen of that same patient's tumor were used as the target and effector cell, respectively. We demonstrate that the CTLs were capable of selective homing to and killing autologous lung tumors grown either subcutaneously or as experimental metatastases in the lung of SCID mice. We also observed that there was a defined period of time after a single injection of CTLs in which the tumor's growth was inhibited and after this point the tumors regrew. Most importantly, the MHC class I antigen expression on the surviving tumor cells was significantly reduced or absent compared to the original tumor. These data and the use of this model for development of improved adoptive T-cell based immunotherapeutic approaches to cancer are discussed.
Methods
Animals
Six to eight week old male CB17-scid/scid mice used for this study were obtained from Taconic Labs (Germantown, NY). They were housed in microfilter cages (Lab Products, Maywood, NJ) at Roswell Park Cancer Institute. All cages, water, and food (Teklad Mills, Wienfield, NJ) were supplied after autoclaved. The cages were maintained in an air-conditioned and light-controlled (12 h/day) room and all handling and operations were done in a laminar flow hood.
Tumor cells
The cell line LT 391-06 was derived from a solid tumor mass excised from the lung of a patient with large cell lung carcinoma. The tumor was obtained as a portion of a surgical specimen, minced with scalpels and digested for 4 hours at 37°C in AIM-V (GibcoBRL, Carlsbad, CA) containing Dispase 8 ug/ml (GibcoBRL) Collagenase 100 ug/ml (GibcoBRL) and Dnase, 10 Ku/ml (Roche). The resultant cell slurry was passed through a nylon mesh screen to break up clumps and layered over two densities of Lymphoprep (Axis-Shield, Oslo, Norway). 7 ml of 1.077 density Lymphoprep was added to a 15 ml conical centrifuge tube followed by 7 ml of a 75% Lymphoprep, 25% 1 × PBS mixture. 10 ml of the cell slurry was layered atop the Lymphoprep and was separated by density centrifugation. The layers were collected sequentially with the top layer containing the majority of tumor cells and the lower layer containing the majority of TIL. Tumor was resuspended and cultured in a mixture of 50% F12 nutrient mixture (GibcoBRL) 50% Basal Medium Eagle (GibcoBRL) 10% FBS (HyClone, Logan, UT) 100 u/ml Penicillin, 100 ug/ml streptomycin, 2 mM L-glutamine (GibcoBRL) 5 × 105 β-mercaptoethanol. Tumor cells were incubated at 37°C and carefully monitored for growth and expansion. Immunohistochemistry (IHC) was performed by Phenopath Laboratories, Seattle, WA in order to confirm that expanding cell lines indeed arose from a lung carcinoma. This tumor cell line expresses HLA-A11, B44 and Cw1203. The lung tumor cell line 936T derived from a surgically removed squamous lung carcinoma lesion and the lung tumor cell line 81-86T derived from a surgically removed large cell carcinoma lesion were kindly provided by Dr. Jill Siegfried of the University of Pittsburgh, Pittsburgh, PA. The 936T cell line expresses the same tumor antigen that is expressed by the LT-391 06 cell line, and expresses HLA-Cw1203 antigen. The cell line 81–86 expresses the tumor antigen but has a HLA phenotype different from that of the cell line LT391-06. 81-86-1203 is the lung tumor cell line 81–86 transfected with HLA-Cw1203 used for positive control. HLA-Cw1203 stable transfectants of tumor cells were generated by retroviral transfection resistant to blasticidin enabling drug selection. A549 is an allogeneic bronchioalveolar carcinoma cell line purchased from ATCC (Cat# CCL 185). This tumor cell line is antigen negative and expresses HLA B44 and Cw1203 antigens. HLA typing of tumor cells was performed at the Puget Sound Blood Center in Seattle, WA. Tumor cells were maintained at 37°C in a 5% CO2 atmosphere in BME/Nutrient mixture F-12 (Life Technologies, Inc., Grand Island, NY), supplemented with 5% heat-inactivated FBS. Cells were allowed to grow to 80% confluency and were harvested with 0.25% typsin-1 mM EDTA in HBSS.
Cytotoxic T lymphocytes
Tumor infiltrating lymphocytes (TIL) that the CTLs derived from were expanded post extraction from the tumor matrix (described above) and cultured in RPMI 1640 medium (GibcoBRL)containing 10% heat inactivated human serum obtained from an in house donor pool, 100 u/ml Penicillin, 100 ug/ml streptomycin, 2 mM L-glutamine (GibcoBRL). The media was supplemented with 100 u/ml Proleukin IL-2 from (Chiron Therapeutics, Seattle, WA) and 30 ng/ml Orthoclone OKT3 (Ortho Biotech). Rapidly growing T cells were split upon confluence and re-fed with fresh media containing a final concentration of 100 u/ml IL-2 and 30 ng/ml mAb OKT3. Tumor antigen-specific T cell lines were obtained by stimulating 1.5 × 106 T-cells with 1 × 105 gamma irradiated (20000 rads) autologous tumor cells over a 7-day cycle. The stimulations were performed in 24 well plates, in 2 ml CTL medium, containing 25 μ/ml IL-2, 10 ng/ml IL-7 (Endogen, Woburn, MA). Media was replaced every 2 – 3 days with IL-2 and IL-7. T cell lines were tested for specificity in a chromium release assay. T cell lines with specific cytotoxicity were cloned by limiting dilution. Per well, T cells, 1 × 104 allogeneic irradiated LCL (8000 rads), 7.5 × 104 allogeneic irradiated PBL (4000 rads) 50 u/ml IL-2 and 30 ng/ml mAb OKT3, 200 ul CTL media per well. Growth positive wells were tested for specificity in a 5 hour chromium release assay. Responding wells were expanded in T25 flasks containing 20 ml CTL media, 50 μ/ml IL-2, 30 ng/ml mAb OKT3, 25 × 106 irradiated allogeneic PBMC (4000 rads), 5 × 106 allogeneic LCL (8000 rads) and approximately 2 × 105 of the specific clone. V-beta analysis performed upon isolated clones by flow cytometry confirmed the homogeneity of the clone. Expanded cells were viably frozen in 90% Fetal Calf Serum (HyClone, Logan, UT) and 10% DMSO (Sigma Chemical co, St Louis, MO). Cells were thawed and rested 24 hours in CTL media containing 25 μ/ml IL-2 prior to each use. Anti-LT391-06 CTL is tumor antigen specific CD8+ T cell clone derived from TILs of lung tumor patient number LT391-06. This clone recognizes a unique shared antigen restricted by HLA-Cw1203 antigen. Studies to identify more precisely this antigen are currently in progress. A CD8+ T cell clone which recognizes an influenza nucleoprotein specific antigen restricted by HLA-B27 antigen was used as a control. Cells were cultured at 37°C in a 5% CO2 atmosphere in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated FBS.
Antibodies
Antibodies for phenotyping the cell surface of CTLs included mouse anti human CD2, CD3, CD4, CD8, CD11a (LFA-1), CD25 (IL-2Rα), CD27, CD28, CD29 (β1-integrin), CD44, CD45RO, CD45RA, CD49a (α1 integrin), CD49b (α2 integrin), CD49d (α4 integrin), CD49e (α5 integrin), CD49f (α6 integrin), β7-integrin, CD54 (ICAM), CD56 (NCAM), CD62L (L-selectin), CD69, CD95 (Fas), CD95L (FasL), CD122 (IL-2Rβ), CD132 (IL-2Rγc), CD134 (OX40), CD152 (CTLA-4), CD154 (CD40L), CD161 (NK1.1), IL-6R, IL-9R, IL-10R, CCR5, CCR7, CXCR3 and MHC Class 1 antigens. All these antibodies were purchased from BD-Pharmingen (San Diego, CA). The mAb HC10 which recognizes a determinant expressed on all β
2-microglobulin-free HLA-B and HLA-C heavy chains, as well as on β
2-microglobulin-free HLA-A10, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, and HLA-A33 heavy chains was developed and characterized as described [
39,
40].
Flow cytometry
Single color staining was performed by incubating 5 × 105 viable cells with an optimal amount of fluorescence-coupled antibodies. After several washes, approximately 10,000 cells per sample were analyzed on a FACScan (Beckton Dickinson, San Jose, CA).
Cytokine measurement
Supernatant cytokine release was determined by sandwich ELISA. Serial dilutions of samples were analyzed for IFN-γ and TNF-α. Antibodies were purchased from BD-Pharmingen.
Chromium release assay
Tumor cells were labeled with 3.7 MBq (100 μci) 51Cr for 1 hr at 37°C. Tumor cells were washed and re-suspended in RPMI 1640 medium supplemented with 10% FBS. After counting, CTLs were placed in 96 well plates. Subsequently 51Cr labeled tumors cells were added to appropriate wells. Following a 5 hr incubation at 37°C in a 5% CO2 atmosphere, plates were centrifuged at 400 rpm for 5 min. The supernatant of each well was collected individually and the 51Cr release was assessed in a gamma counter. The percentage of specific 51Cr release was calculated using the standard formula: %51Cr release = [(51Cr release of samples-spontaneous 51Cr release)/(maximum 51Cr release-spontaneous 51Cr release)] ×100. All samples were run in quadruplicate in two different sets of experiments.
Tumor growth
One hundred μl of a lung tumor cell suspension (at a concentration of 5 × 106/ml) were injected subcutaneously or intravenously into SCID mice. Subcutaneously grown tumors were measured with a venier caliper to determine the shortest diameter (A) and the longest diameter (B). The volume was then calculated by using the formula V = (A2B)/2. For intravenously grown tumor, lung were excised and weighed.
Depletion of murine NK cells
To deplete NK cell activity, 24 h before injection of tumor cells, we injected every 4 weeks i.p 100 μl of TMβ-1 antibody obtained as ascites fluid from a hybridoma kindly provided to Dr. Richard Bankert (State University of New York at Buffalo, Buffalo, NY) by Dr. T. Tanaka (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). This antibody blocks murine IL-2β receptor and exhibits no crossreactivity with human IL-2β receptor [
41].
IL-2 treatment
Subcutaneous tumor bearing SCID mice, previously treated with TM-β1 antibody, received daily subcutaneous injections of 20,000 IU of Proleukin IL-2 (Chiron Therapeutics, Seattle, WA) starting the day of adoptive transfer of CTLs.
Histopathology
After animals were sacrificed, tumor and lung or other organs were excised for histological analysis. Excised tissues were fixed for 24 h in 10% buffered formalin. The tissues were then dehydrated for 18 h by an automatic tissue processor and embedded in paraffin blocks. Five-micrometer paraffin tissue sections were cut and the tissue sections were stained with hematoxylin and eosin.
Immunohistochemical analysis
Immunohistochemical analysis was performed according to the staining procedure for paraffin sections provided by Vector Laboratories (Burlingame, CA). Briefly, paraffin sections (5 μm) were deparaffinized and rehydrated through xylene and alcohol series. The tissue sections were then incubated with 0.3% H2O2 in methanol for 30 min to quench endogenous peroxidase activity and then incubated with diluted normal goat serum for 20 min to block nonspecific bindings. The sections were then incubated at room temperature for an hour with anti-HLA class I heavy chain mAb HC-10 or CD8 (Novocastra Laboratories Ltd., Newcastle, UK). Biotinylated goat anti-mouse IgG antibodies (at a1:500 dilution) were then applied to tissue sections for 30 min at room temperature. Vectorastain ABC kit (Vector Laboratories, Burlingame, CA) was used for signal enhancement and peroxidase substrate DAB was used for color development.
RT-PCR
Total RNA was extracted from lung and liver of SCID mice baring tumor and/or CTL and RT-PCR was done as per manufacturer's instructions (Superscript Preamplification System; Life Technologies, Inc). Primers for human CD8 were: 5'-TTTAGCCTCCCCCTTTGTAAAACGGGCG-3' and 5'-TTTCGGCGAGATACGTCTAACCCTGTGC-3'. Primers for human IFN-γ were: 5'-CTCCTTTTTCGCTTCCCTGTTTTAGCTGCTGG-3' and 5'-GCATCGTTTTGGGTTCTCTTGGCTGTTACTGC-3'. Primers for β-actin were: 5'-ATCTGGCACACCTTCTACAATGAGCTGCG-3' and 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3'. Amplification was done for 35 cycles with denaturing at 94°C for 30 s, annealing at 60°C for 1.5 min, and extension at 72°C for 1.5 min.
Statistical analysis
Statistical analysis was conducted using two methods. For lung weights we used non-parametrical unpaired Student t-test analysis. For analysis of sc models we took the reference as the day for tumors to reach 500 mm3 in volume using the long rank test provided in the Graphpad Prism Software Package version 4.01 (Graphpad, San Diego, CA). P values < 0.05 referred as statistically significant.
Discussion
Despite theoretical appeal and actual data regarding the efficacy of T cell-mediated immunotherapy, only a small subset of human cancer patients who present with tumors actually experience clinically relevant benefits. The results of our studies presented here indicate that tumor antigen-specific, HLA-Cw1203 restricted CTLs, generated from the cells that infiltrate a patient's lung tumor, are able to lyse autologous lung tumor cells
in vitro and in a SCID mouse model. Consistent with these findings, in humans the
in vitro generation and expansion of autologous CTLs that recognize lung [
1‐
5], melanoma [
25‐
27], renal [
28], ovarian [
29], leukemia [
30‐
32], lymphoma [
33,
34] and breast [
35‐
38] tumor antigens have been reported. Furthermore, studies using patients' melanoma tumors [
42,
43] or revealed that melanoma antigen-specific CTLs killed allogeneic melanoma cells which shared the HLA class I restricting element, to a similar extent as autologous melanoma cells. Other studies showed that CTLs can be effective cytotoxic effector cells for adoptive immunotherapy [
19‐
24] as well.
Phenotypic analysis has often been utilized to characterize the differentiation, proliferation, activation status and immune response of a particular immune cell. Since there was no prior information about the phenotypic characteristics of lung tumor derived, tumor antigen-specific CD8
+ CTLs, we needed to first characterize the cell surface markers of the patient derived and
in vitro-expanded CTLs. A previous study, which investigated the functions of CD8
+ T cell CD27
+CD45RA
+ subsets, suggested that CD27
+CD45RA
+CD8
+ T cells are naïve T cells, while CD27
+CD45RA
-CD8
+, CD27
-CD45RA
-CD8
+, and CD27
-CD45RA
+CD8
+ T cells are memory, memory/effector and effector cells, respectively [
44]. Classification is made on virus epitope specific CD8+ T cells based upon CCR5 and CCR7 expression indicates that CCR5 expression is restricted to memory and effector cells, whereas CCR7 is expressed only in naïve and memory cells [
45]. The information we gathered after phenotypic analysis of CTLs revealed that these CD8
+ T cells were CD25
+CD27
-CD45RA
-CD45RO
+CD44
+CD69
+ CCR5
+CCR7
+, and based on the previous studies [
45] we can conclude that these cells have an activated memory phenotype, again suggesting that within the patient, positive immune stimulation took place at some point in the course of the disease. Yet, for many patients, and for reasons which are not yet clear, these cells fail to control tumor growth.
It has been difficult to study the antitumor efficacy of human immune effector cells, such as cytolytic T lymphocytes (CTLs) in the clinical setting of lung cancer and there has been little information derived from animal models in this regard and the models that have been developed utilizes subcutaneously grown tumors [
5,
46]. SCID mice that lack functional T and B cells have been shown to be useful hosts to grow human tumors, both from established cell lines and from tumors obtained directly from patients [
46‐
53]. Thus, they may provide an environment for studying the interactions between human tumor cells and autologous tumor antigen-specific CTL. In evaluating the efficacy of these tumor antigen-specific CTLs
in vivo by adoptively transferring them to SCID mice, we found that these CTLs possessed remarkable efficiency at homing to and remaining in the lung bearing autologous tumors (orthotopically grown tumors). Notably, they did not persist in non-tumor bearing lungs or liver. Most importantly, a single injection of these cells, with no further supportive treatments with IL-2 resulted in significant tumor growth suppression in both subcutaneously grown tumor and lung tumor metastasis. Future studies should evaluate other cytokines such as IL-15 in terms of improving CTL effectiveness.
One possibility for improvement of the response may be related to timing of immune treatment since based on our results, the scheduling of CTL injection was found to be very critical in delaying tumor growth. If tumor antigen-specific CTLs are generated early enough they may be more effective in controlling tumor growth. Another obvious reason for incomplete cure could be the limited survival of CTLs in vivo. Improving the survival of CTLs in this setting remains for further investigation. The number of CTLs is another issue that needs to be considered; in all our experiments we administered a maximum dose of 5 × 106 CTLs. In future experiments, it is essential to know whether the tumor growth will be further inhibited by using a higher dose of CTLs. Notably however, administration of CTLs at consecutive time points did not significantly enhance the antitumor effect compared to a single injection of CTLs at an early time point. This may be due to the fact that the first injection resulted in the majority of the effect and that possible tumor cell phenotype changes occurring after the first CTL treatment provided tumor cells with escape mechanisms from recognition and destruction by subsequently administered CTLs.
Any effective CD8+ T cell based-immunotherapy depends on the continued HLA class I antigen expression by tumor cells [
7,
54]. Although loss of HLA class I antigen expression is previously described in patient tumors [
7], this study is the first study to show patients' HLA Class I positive lung tumor cells following tumor antigen-specific CTL treatment showed marked decreased HLA class I expression using this mouse model. Whether this change represents a general phenomenon is not known at present. Nevertheless our results suggest that the application of T cell selective pressure to a tumor cell population favours the outgrowth of tumor cells with defects in HLA class I antigen expression. At this point, we cannot distinguish between selection and ongoing mutation as the major mechanism for this effect. However, Kedl et al [
55] have shown down-modulating peptide MHC class I complexes on APCs by high affinity T cells
in vivo. Since we were still able to detect cytoplasmic but not membrane staining with anti-HLA class I mAb in some of the tumor cells, it is possible that a similar phenomena might be occurring after tumor antigen-specific CTL interaction with autologous tumor cells. In this study, we have not been able to look at changes in tumor antigen expression because we are still in the process of characterizing tumor antigen-specific antibodies. The mechanism(s) underlying decreased HLA class I expression on lung tumor cells is not known. The phenotype of the lung tumor cells we have analyzed in conjunction with the described high frequency of antigen processing machinery abnormalities in tumor cells suggest that defects in this machinery may underlie the HLA class I antigen downregulation we have detected in tumor cells following treatment with tumor antigen-specific CTL. If our hypothesis is correct, the defects in the antigen processing machinery which result in defective loading of HLA class I antigens with peptides and in their instability on the cell membrane may be corrected by IFN-γ. Another explanation of decreased MHC class I expression can be the selection of MHC class I negative variants of the tumor. The concept of selection for tumor cells or shaping the tumor immunogenicity has been previously described [
56]. Using this model, ways could be tested to evaluate the ability of various adjuvants for their ability to increase expression of MHC class I. For example, tumor cells have been shown to upregulate their MHC class I expression after IFN-γ treatment [
56‐
58]. An alternative strategy could rely on the enhancement of the lytic activity of natural killer (NK) cells, since HLA class I antigen downregulation and loss enhance susceptibility of target cells to NK cell-mediated lysis.